In the previous grant period, we characterized the Jun activation domain-binding protein (JAB1), which interacts with and transactivates c-Jun and promotes cellular proliferation. JAB1 was also found as a key negative regulator that controls the activity of p27, a cyclin-dependent kinase inhibitor, by facilitating its relocalization from the nucleus to the cytoplasm and therefore promoting p27 degradation and cell cycle progression. Reduced p27 levels correlate with poor prognosis in a wide spectrum of human tumors and can accelerate tumorigenesis in mouse tissues. Additional data from our laboratory suggest that constitutive transcriptional activation of the Jab1 gene is responsible for JAB1 protein overexpression. Also, Jab1 gene amplification and protein overexpression are associated with low p27 levels and a poor prognosis in breast carcinoma patients. Emerging evidence has indicated that abnormal expression of Jab1 is implicated in the pathogenesis of a variety of cancers. Conversely, targeted silencing of JAB1 increases p27 protein levels, reinstates a G1 checkpoint, dramatically reduces the expression of many cell cycle genes, and induces cell cycle arrest and apoptosis in tumor cells. Although it is assumed that the phenotypes resulting from Jab1 silencing are due to p27 stabilization, this has not been proven. In addition, recent findings from a Jab1 knockout mouse study clearly demonstrated that p27 is elevated as p53 and cyclin E, in Jab-/- embryos resulting in impaired proliferation and accelerated apoptosis. Thus, providing evidence that JAB1 is a critical regulator of p27 degradation. We have mapped the domains within JAB1 that transactivate c-Jun and potentiate p27 degradation. Exactly how those separated activities of JAB1 (e.g. AP-1 co-activator and negative regulator of p27 cell cycle inhibitor) work together and contribute to breast tumorigenesis is not clear. In this second funding period, we propose to test the hypothesis that JAB1 is an important mediator of cell proliferation and that its aberrant expression is oncogenic and contributes to the progression of some breast carcinomas. With this continuation application, we propose to identify the oncogenic function of JAB1 by a series of molecular approaches. We will then evaluate whether JAB1 can represent an attractive target for therapeutic inhibition in breast cancer and investigate its role in resistance to treatment with Herceptin. Finally, we will characterize the control Jab1 expression in human breast carcinoma by identifying the transcriptional positive and negative regulators of Jab1, thus will indicate the upstream pathways responsible for its increased JAB1 levels in tumor cells and not in non-transformed cells. Our goals are to understand the effective transcriptional up-regulation of the Jab1 gene in breast tumors and to find a more effective therapeutic treatment for breast cancer. A more complete knowledge of JAB1's oncogenic function could be of therapeutic importance for other solid tumors with a similar phenotype. Upstream targets regulating JAB1 expression may be identified for use in future therapeutic paradigms.